The focus of this research is to determine how a single population of ion channels, gated by cGMP, transduces the photochemistry of the outer segment into a current that conveys all pre-processed visual information. The current through these channels is generated by the influx of both Na and Ca ions. Since changes in cytosolic Ca levels are required to adjust the sensitivity of photoreceptors to changes in background light levels, a fundamental question about phototransduction is what fraction of the outer segment current is carried by Ca. The channel could either maintain fixed influx ratios of Na and Ca or alter the ratio in response to varying levels of background light via changes in cytosolic cGMP or Ca levels. Thus, one aim of our research is to determine the influx ratios. Because the ions cannot be distinguished electrophysiologically, we will measure 22Na and 45Ca influx in cultured mammalian cells that transiently express the channels. Continuous surveillance of cytosolic cGMP levels through binding sites on the surface of the channel results in alterations of the probability of channel opening. Another fundamental question, therefore, is how the protein monitors these concentration changes and subsequently converts the information into an conductance change. By expressing the cDNA for the channel at low levels in oocytes, we will record from single channel patches and examine the relationship between occupancy of each cGMP binding site with channel opening. Finally, to explain the molecular basis of cGMP binding specificity and divalent interactions with the channel, we will examine nucleotide-activated currents in mutant forms of the channel. Alterations of the amino acid side chains that interact with cGMP might provide clues to the nucleotide specificity, while alterations in putative Ca binding Glu and Asp residues might be useful in defining the channel topology.